Molecular Analysis Of Leukocyte Activation By Chemoattractants

本项目的目的是确定血液白细胞迁移到炎症或感染的特定组织部位的分子机制和生物学背景。我们关注的是介导这一过程的化学引诱蛋白，并已经确定了部署在白细胞细胞表面的化学引诱受体大家族的成员。我们还发现了由病毒（包括疱疹病毒、痘病毒和艾滋病毒）制造的多种化学引诱剂和化学引诱剂受体模拟物的成员。我们将基因组学、分子生物学、细胞生物学和流行病学作为分析这些分子的主要方法。一个主要目标是确定个体化学引诱剂和化学引诱剂受体的特定疾病关联，以确定潜在的新治疗靶点。一个关键的策略是分析疾病模型中基因敲除小鼠的表型，以及人类疾病队列中相应人类基因功能突变丧失的关联。2015财年，我们报告了以下领域的发现：动脉粥样硬化;2. 3.老年痴呆症；原发性免疫缺陷疾病WHIM综合征；同种异体移植物耐受机制。

The aim of this project is to define the molecular mechanisms and biological contexts for blood leukocyte migration to specific tissue sites that are inflamed or infected. We have focused on chemoattractant proteins that mediate this process and have identified members of a large family of chemoattractant receptors that are deployed on the leukocyte cell surface. We have also identified members of a diverse group of chemoattractant and chemoattractant receptor mimics made by viruses, including herpesviruses, poxviruses and HIV. We use genomics, molecular biology, cell biology and epidemiology as the principle methods for analyzing these molecules. A major goal is to identify specific disease associations of individual chemoattractants and chemoattractant receptors, in order to identify potential new therapeutic targets. A key strategy is to analyze phenotypes of gene knockout mice in disease models as well as associations of loss of function mutations in the corresponding human genes in human disease cohorts. In FY15 we reported discoveries in the following areas: 1. atherosclerosis; 2. Alzheimer's Disese, 3. The primary immunodeficiency disease WHIM syndrome, and 4. mechanisms of allograft tolerance. 1.) In FY16, we developed the first knockout mouse for the CXC chemokine receptor Cxcr1. Unlike Cxcr2, which has been well-studied, Cxcr1, despite being closely related had remained an enigma. Our development of this mouse provided an opportunity to interrogate its biological functions which we did in collaboration with two groups focused on infectious disease pathogenesis. The first study related to Candida albicans pathogenesis was performed by M. Lionakis starting when he was in the LMI as an ACI and completed after obtaining an independent position in LCID. The work showed that the receptor is important for control of systemic candidiasis operating at the level of control of candidicidal mechanisms in neutrophils, not at the level of neutrophil trafficking to kidney. This paradigm was supported by a second collaborative study examining the knockout mice in a model of Pseudomonas infection of the lung. Together, the studies establish Cxcr1 for the first time as a key receptor in acute infectious disease pathogenesis by regulating neutrophil activation. 2.) In FY16, we reported the case of a patient with WHIM syndrome, a rare immunodeficiency disorder, caused by a novel CXCR4 mutation previously associated with Waldenstrom's Macroglobulinemia. The mutation affects the receptor in the same way as previously described WHIM mutations. Interestingly, the same frameshift occurs in the WHIM patient's receptor as in the Waldenstrom's patient's receptor but by distinct genetic mechanisms. This work provides further evidence that the C-tail of CXCR4 is a genetic hotspot for mutation. 3.) In FY16 we also reported that pre-treatment of allogeneic bone marrow recipients with the macrophage depleting agent clodronate durably enhances hematopoietic chimerism and promotes donor-specific skin allograft tolerance. Donor specific allograft tolerance is a holy grail in transplantation that, if achieved, could massively expand the number of potential organ donors and reduce the toxicity from immunosuppression associated with the procedure. It's known that establishing hematopoietic chimerism with the donor can facilitate donor-specific organ allograft tolerance. It's also thought that engraftment of stem cells requires vacant bone marrow niches. We therefore mobilized stem cells by depleting bone marrow macrophages with clodronate in the recipient hoping to enhance donor engraftment and chimerism and hence skin allograft tolerance. We observed durable enhancement of chimerism in blood, spleen and bone marrow out to over 200 days. We have used this proof of principle finding to move on now to other niche vacating strategies. 4.) In FY16, we showed that following control of acute infection of mice with the myotropic Colombiana strain of Trypanosoma cruzi, parasites persisted in tissue at low levels associated with development of systemic necrotizing vasculitis. Infectious agents are often considered potential triggers for chronic inflammatory disease, including autoimmunity; however, direct evidence is usually lacking. In our study, lesions occurred in many but not all organs and tissues, with skeletal muscle arteries most severely affected, associated with myositis, atrophy, paresis/paralysis and death. Histopathology showed fibrinoid vascular necrosis, rare amastigote nests within skeletal muscle myocytes, and massive leukocyte infiltrates composed mainly of inflammatory monocytes, F4/80+ macrophages and T. cruzi tetramer-specific CD8+ T lymphocytes capable of producing IFN and TNF, but not IL-17. T. cruzi-specific IgG was detected in serum from infected mice, but antibody deposits and neutrophilic inflammation were not features of the lesions. Thus, T. cruzi infection of mice may be a specific infectious trigger of paralyzing systemic necrotizing vasculitis most severely affecting skeletal muscle, driven by pathogen-specific Type I immune responses.